Many assays exist for detecting and measuring analytes of small quantity in the presence of a large volume of other substances. Such assays typically make use of the high binding affinity between the analyte (the substance to be detected or measured) and a second molecule having a high degree of specificity for binding to that analyte. These assays are often referred to as ligand-binding assays.
One of the most common ligand-binding assays are immunoassays. Immunoassays typically employ an antigen and an antibody which specifically binds to the antigen to form an antibody/antigen complex. In order to measure the extent of the antibody/antigen binding, one member of the complex is generally labeled or tagged with a traceable substance. The presence of the traceable substance, and hence the presence of the antibody or antigen to which it is attached, may then be detected or measured using a variety of different techniques depending upon the unique characteristics of the label employed. These techniques may include an analysis and measurement of scintillation counting, fluorescence, absorption, electrochemistry, chemiluminescence, Rayleigh scattering and Raman scattering. Of these techniques, fluorescence spectroscopy has been one of the most widely used readout methods, primarily because of its high sensitivity.
Although fluorescence spectroscopy has seen substantial use in scientific research and clinical diagnostics, there are disadvantages in using fluorescence spectroscopy. For instance, the different types of fluorescent molecules used in fluorescence spectroscopy typically require excitation with photons of differing wavelengths. Therefore, if the detection of multiple fluorescent molecules is desired in a single sample, multiple light sources may be required. If a single light source is used, there will often exist a spectral overlap between the emission of the different fluorescent molecules such that reliable individual and quantitative detection of multiple analytes in a single sample is limited.
Today, many assays require the concomitant determination of more than one analyte in a single test sample (e.g., the screening of cancer markers, such as α-fetoprotein and carcinoembryonic antigen). There are two general approaches to assaying multiple analytes in a single sample. One approach immobilizes different binding molecules on a solid support at spatially separated addresses. Multiple analytes can then be detected using the same label, with identification based on address location. Alternatively, different labels can be used to detect multiple analytes simultaneously in the same spatial area. In this case, each analyte obtains its own distinct label.
We have explored Raman spectroscopy as an alternative to fluorescence spectroscopy. Raman spectroscopy measures the level of Raman scattering induced by the application of a radiation source, i.e. light source, on an analyte. The light incident on the analyte is scattered due to excitation of electrons in the analyte. “Raman” scattering occurs when the excited electron returns to an energy level other than that from which it came, resulting in a change in the wavelength of the scattered light and giving rise to a series of spectral lines at both higher and lower frequencies than that of the incident light. The series of spectral lines is generally called the Raman spectrum.
Conventional Raman spectroscopy usually lacks sufficient sensitivity for use as a readout method for immunoassays. Raman spectroscopy is also unsuccessful for fluorescent materials due to the broad fluorescence emission bands tend to swamp the weaker Raman bands.
However, a modified form of Raman spectroscopy based on “surface enhanced” Raman scattering (SERS) has proved to be more sensitive and thus of more general use. In the SERS form of Raman spectroscopy, the analyte whose spectrum is being recorded is closely associated with a roughened metal surface. This close association leads to a large increase in detection sensitivity, the effect being greater the closer the analyte sits to the metal surface.
The manner in which surface enhancement occurs is not yet fully understood, but it is thought that the incident light excites conduction electrons in roughened metal surfaces or particles, generating a plasma resonance (plasmon). As a result, the electromagnetic field in the vicinity of the metal surface is greatly amplified, giving rise to enhanced Raman scattering in molecules located close to the surface.
Surprisingly, there have been only a few reports on the application of SERS for detection in immunoassays. Two of these approaches used a sandwich-type assay, which coupled surface and resonance enhancements. In particular, Rohr et al., Anal. Biochem. 1989, 182, 388, used labeled detection antibodies and roughened silver films coated with a capture antibody (see also U.S. Pat. No. 5,266,498 to Tarch et al.), and Dou et al., Anal. Chem. 1997, 69, 1492, exploited the adsorption on silver colloids of an enymatically amplified product. Another approach by White et al., International Application Publication No. WO 99/44065, employs an immunoassay based on the displacement of SERS and surface enhanced resonance Raman (SERRS) active analyte analogs which are modified so as to have particular SERS and SERRS surface seeking properties. Upon introduction of a sample, the analyte analogs are displaced by the analyte of interest in the sample and exposed to a SERS or SERRS surface, such as an etched or roughened surface, a metal sol or an aggregation of metal colloid particles. Raman spectroscopy is then performed to detect the displaced analyte analog associated with the SERS or SERRS surface to determine the presence or quantity of the analyte in the sample.
A major barrier that prohibits using SERS for the direct detection of biological samples is that the surface enhancement effect diminishes rapidly with increasing distance from the metallic surfaces. In other words, strong SERS signals are observed only if the scattering centers are brought into close proximity (<100 nm) to the surface. In addition, although Raman spectra of biomolecules can be obtained on silver surfaces when coupling SERS and resonance enhanced scattering, the spectra are usually lacking of sufficient chemical content and/or signal amplitude to be used for immunoassay purposes.
We have overcome these barriers by developing a novel class of Raman-active reagents having both Raman-active reporter molecules and binding molecules integrated with each other on the same SERS surface. In each of the above systems, the SERS or SERRS surface and the Raman-active molecule are not integrated with each other, but are merely placed in close proximity to each other by the combination of an analyte sandwiched between an antibody immobilized on the enhancing surface and an antibody attached to a Raman-active molecule, or the combination of the SERS or SERRS surface with a particular SERS or SERRS surface seeking group coupled to an analyte analog and a Raman-active molecule, after exposure to the sample.